Dengue virus rpa-crispr-cas12b detection system and detection method thereof

Abstract

The application belongs to the technical field of dengue virus detection, and specifically discloses a dengue virus RPA-CRISPR-Cas12b detection system and a detection method thereof. The application selects a dengue virus conservative region as an amplification and detection target, provides a specific sgRNA (the sequence is shown as SEQ ID NO. 6) and an RPA primer for detecting dengue virus; the RPA primer is used for RPA amplification on a to-be-tested sample, a specific sgRNA is used for CRISPR reaction on an amplification product, and whether the to-be-tested sample contains dengue virus can be judged through color change of a reaction system. The application provides corresponding sgRNA and RPA primer sequences and applications, and the provided RPA-CRISPR-Cas12b detection system and detection method have technical advantages of simple operation, rapid and sensitive reaction, strong specificity and the like, and meet application requirements of on-site and instant visual detection of dengue virus.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of dengue virus detection, and particularly relates to a dengue virus RPA-CRISPR-Cas12b detection system and a detection method thereof. BACKGROUND

[0002] Dengue virus (DENV) infection has a great health impact in the world's tropical and subtropical countries. The increase in infection rate greatly increases the morbidity and mortality, most commonly caused by dengue hemorrhagic fever and dengue shock syndrome. Although developing an effective and durable vaccine has been the main goal of controlling and preventing DENV infection, the currently licensed vaccines have limitations. Therefore, early detection of DENV is crucial for implementing preventive measures for DENV infection.

[0003] Traditional methods for detecting DENV include reverse transcription polymerase chain reaction (RT-PCR), enzyme-linked immunosorbent assay (ELISA), serology, etc., however, these methods are highly dependent on specific DENV antibodies, complex equipment, operating levels, high cost and are susceptible to cross-reactions, and are also limited in use scenarios. Therefore, there is an urgent need to establish a detection system with triple breakthroughs in sensitivity, specificity and rapidity, especially suitable for on-site screening at the early stage of a virus outbreak, hierarchical diagnosis and treatment at primary medical institutions, and travel health monitoring.

[0004] Nucleic acid isothermal amplification technology does not require repeated thermal denaturation, does not require special instruments, and has faster reaction speed, and is suitable for on-site rapid detection, and has been widely used in life science research and related fields. At present, there are more than 10 kinds of nucleic acid isothermal amplification technologies, among which RPA (recombinase polymerase amplification) is an isothermal nucleic acid amplification technology that can rapidly (10-20 minutes) amplify target nucleic acid sequences at 37-42℃, and has broad application prospects in many fields such as disease diagnosis and pathogen identification. The CRISPR / Cas system has specific gene targeting ability and has become a new diagnostic platform in recent years, and is widely used in the detection of pathogens, free tumor DNA, methylation, etc. due to its high sensitivity and specificity. Cas12b protein, under the guidance of sgRNA, activates its transcleavage activity when recognizing the matching target sequence, i.e. cutting single-stranded non-specific nucleic acid sequences indiscriminately. SUMMARY

[0005] In view of the deficiencies in the prior art, the application provides a dengue virus RPA-CRISPR-Cas12b detection system and a detection method thereof.

[0006] In order to achieve the above-mentioned purpose, the application provides the following technical solutions:

[0007] The application selects a conserved region of dengue virus as an amplification and detection target, takes DV dengue virus ID: MW512436.1 (10501-10723) as a target detection region, and the nucleotide sequence of the gene is shown as SEQ ID NO. 1.

[0008] In a first aspect, the application provides an sgRNA probe for detecting dengue virus, wherein the sgRNA comprises a specific target sequence shown as SEQ ID NO. 2.

[0009] Further, the nucleotide sequence of the sgRNA is shown as SEQ ID NO. 6.

[0010] In a second aspect, the application provides the use of the above-mentioned sgRNA probe in the preparation of a kit for detecting dengue virus.

[0011] In a third aspect, the application provides a dengue virus RPA-CRISPR / Cas12b detection system, comprising an RPA amplification reaction system and a Cas12b detection reaction system; the Cas12b detection reaction system comprises an sgRNA shown as SEQ ID NO. 6.

[0012] Further, the RPA amplification reaction system comprises an RPA primer pair shown as SEQ ID NO. 4-5.

[0013] In a fourth aspect, the application provides a kit for visually detecting dengue virus, comprising an sgRNA shown as SEQ ID NO. 6 and an RPA primer pair shown as SEQ ID NO. 4-5.

[0014] Further, the kit further comprises an AaCas12b enzyme protein, a 10x AaCas12b Buffer, an ssDNA reporter molecule, and RNase free ddH2O (nuclease-free water).

[0015] Further, the ssDNA reporter molecule is an FAM-BQ1 labeled ssDNA: FAM-TTTTTTT-BQ1.

[0016] The application also provides the use of the above-mentioned kit in the preparation of a dengue virus detection reagent.

[0017] In a fifth aspect, the present application provides a RPA-CRISPR / Cas12b-based dengue virus visual detection method, comprising the following steps: first, performing RPA amplification on the sample to be tested, and the nucleotide sequence of the RPA primer pair used is shown in SEQ ID NO. 4-5; then performing CRISPR reaction on the RPA amplification product using a Cas12b detection reaction system, after the CRISPR reaction is completed, observing the color change of the reaction system can determine whether the sample to be tested contains dengue virus; the Cas12b detection reaction system comprises sgRNA with a sequence shown in SEQ ID NO. 6, AaCas12b enzyme protein, 10x AaCas12b Buffer, ssDNA reporter molecule, and nuclease-free water; the ssDNA reporter molecule (Reporter) is a FAM-BQ1-labeled ssDNA: FAM-TTTTTTT-BQ1.

[0018] Further, in the RPA amplification reaction system in the step, the final concentration of the forward primer and the reverse primer is 640 nM-1 µM, and more preferably 800 nM.

[0019] The amplification reaction conditions are: the temperature is 37-45°C, and the preferred reaction temperature is 43°C; the reaction time is 15-60 min.

[0020] Further, in the Cas12b detection reaction system in the step, the final concentration of the sgRNA, the AaCas12b enzyme protein, and the ssDNA reporter molecule is 5 ng / µL, 0.06 µM, and 2 µM, respectively.

[0021] The CRISPR reaction conditions are: the temperature is 37-45°C, and the preferred reaction temperature is 43°C; the reaction time is 15-60 min.

[0022] Compared with the prior art, the present application has the following beneficial effects:

[0023] The present application takes the dengue virus DV MW512436.1 (10501-10723) gene as the target region, and the designed sgRNA can achieve the detection result within 30-40 minutes without the help of complex instrument equipment when used for detecting dengue virus, and the minimum detection of each reaction can be 1E0 copies / T of sample. The RPA-CRISPR / Cas12b double mechanism has the ability of rapid amplification and high specificity detection, which can exclude cross reaction and avoid false positive. The present application has many technical advantages such as simple operation, rapid reaction, excellent sensitivity and strong specificity, and is especially suitable for screening of early infection (1-2 days before symptoms appear) or asymptomatic carriers, which makes up for the lag of colloidal gold test strip depending on antibody (4-5 days after infection), and meets the application requirements of on-site instant visual detection. BRIEF DESCRIPTION OF DRAWINGS

[0024] Figure 1 DV target sgRNA position diagram designed for the present application.

[0025] Figure 2 The fluorescence intensity determination results of the amplification products of each sgRNA and the corresponding template for the CRISPR reaction of the present application: A: sgRNA screening fluorescence change rate; B: sgRNA screening fluorescence curve; NTC is negative control.

[0026] Figure 3 DV target RPA primer position diagram designed for the present application.

[0027] Figure 4 DV target RPA primer screening results; A: RPA primer screening fluorescence change rate; B: fluorescence curve of different RPA primers under high concentration; C: fluorescence curve of different RPA primers under low concentration; NTC is negative control.

[0028] Figure 5 DV sensitivity verification results; A: fluorescence growth rate per unit time of different template concentration groups; B: fluorescence amplification curve of different template concentration groups; NTC is negative control.

[0029] Figure 6 DV target sensitivity verification results; A: fluorescence change rate in the sensitivity confirmation stage of DV target; B, C: RPA-CRISPR fluorescence curve of DV target under high and low concentrations; NTC is negative control.

[0030] Figure 7 DV target real sample verification results; A: DV real sample verification fluorescence change rate; B: DV target real sample verification fluorescence curve.

[0031] Figure 8 Dengue virus qPCR actual sample verification results.

[0032] Figure 9 DV target specificity verification results; A: DV specificity verification fluorescence rate of change; B: DV target specificity verification fluorescence curve; PTC: No. 2 real sample nucleic acid (type I dg2-157), NTC: negative control.

[0033] Figure 10 Synsor Pocket-One platform positive and negative judgment diagram.

[0034] Figure 11 Synsor Pocket-One platform preliminary test results.

[0035] Figure 12 Synsor Pocket-One platform sensitivity verification results. DETAILED DESCRIPTION

[0036] The technical solutions of the present application will be described clearly and completely below in combination with the embodiments of the present application. Obviously, the described embodiments are only some of the embodiments of the present application, not all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor fall within the scope of protection of the present application.

[0037] The test methods used in the embodiments of the present application are all conventional methods unless otherwise specified. The materials, reagents, etc. used are reagents and materials available from commercial channels unless otherwise specified.

[0038] Example 1 Experimental materials and methods

[0039] 1. Experimental materials

[0040] 1.1. Experimental instruments

[0041] QPCR instrument-SLAN-96P (Shanghai Hongshi Medical Technology Co., Ltd.), PCR instrument (TC-S / 96 / G / H(b)BA) (Hangzhou Bozhi Technology Co., Ltd.), Qubit 3.0 fluorometer (Thermo Fisher Scientific), Nanodrop 2000 ultramicro spectrophotometer (Thermo Fisher Scientific), metal bath (DH100-2) (Hangzhou Ruicheng Instrument Co., Ltd.).

[0042] 1.2. Experimental reagents

[0043] SynSor AaCas 12b (C2c1) (XS-R-002) (Synsorbio), SynSor DNA / RNA Isothermal Rapid Amplification Reagent (XS-R-101) (Synsorbio), SynSor CRISPR ssDNA Reporter (12b-FAM) (XS-R-201) (Synsorbio), SynSor sgRNA (XS-R-301) (Synsorbio), Gold MIX (Green) (TSE101) (Beijing Qikeli Biotechnology Co., Ltd.), and water used are all UltraPure™ Distilled water, Dnase, Rnase, Free (10977-015) (Infinitti (Shanghai) Trading Co., Ltd.).

[0044] The SynSor CRISPR ssDNA Reporter is specifically FAM-BQ1 labeled ssDNA: FAM-TTTTTTT-BQ1.

[0045] 2. Experimental method

[0046] 2.1. Selection of dengue virus detection target and design of sgRNA

[0047] A conserved region of dengue virus is selected as an amplification and detection target. Dengue virus DV MW512436.1 (10501-10723) is selected as the target detection region (SEQ ID NO. 1). In combination with the host background genome that needs to be avoided, the sgRNA is designed according to the PAM position, GC%, internal dimer structure, fragment structure openness, base position preference, specificity, and other angles using a bioinformatics algorithm. The score can be understood as a success probability. Generally, 3 candidate fragments with scores above 40 are retained. If the scores are generally lower than 40, the number of candidate fragments needs to be increased to improve the success probability. If the sequence in the spacer has the risk of destroying the backbone secondary structure, it will be directly excluded from the candidate list to avoid the risk of specificity and sensitivity.

[0048] Based on the above design principles, the sgRNA is designed for each selected region for subsequent synthesis and verification. The spacer sequence of the designed sgRNA is analyzed for coverage and specificity using NCBI primer-BLAST. The comparison results show that the designed sgRNA spacer sequence has a coverage of 100% within the detected species and good inclusivity; the number of base pairs matched across species is <15 nt, and the specificity is good. Under the premise of ensuring specificity, the sgRNA in Table 1 is selected for synthesis (the sgRNA position is shown in Figure 1 ).

[0049] Table 1 sgRNA information used in the present application

[0050]

[0051] * The part marked with a horizontal line at the bottom is the spacer region of sgRNA

[0052] 2.2, sgRNA performance verification

[0053] 2.2.1 PCR primer design

[0054] According to the position of sgRNA design, the region within 100 bp upstream and downstream of sgRNA was selected to design PCR primers (see Table 2). The primers were designed by using common primer design software, and the length of the primers was controlled at 20-25 bp. If there are multiple sgRNA design sites in the same target fragment, the PCR amplification product (the length of the fragment is controlled within 500 bp) will contain all the sgRNA binding sites, so that all sgRNAs can be verified for performance under the same template.

[0055] Table 2 Template amplification specific primers

[0056]

[0057] 2.2.3 Template amplification verification

[0058] PCR amplification was performed using template DNA and corresponding primers to obtain high-concentration PCR products. The specific amplification system is shown in Table 3, and the corresponding PCR reaction program is shown in Table 4.

[0059] Table 3 PCR system

[0060]

[0061] Table 4 PCR program

[0062]

[0063] 2.2.4 sgRNA verification

[0064] The CRISPR system was prepared according to the system in Table 5.

[0065] Table 5 CRISPR system preparation

[0066]

[0067] Take 1 μL PCR amplification product and mix with the above CRISPR system, set 43 ℃ reaction for 15 min in qPCR instrument, collect FAM fluorescence signal every minute. According to the curve change of fluorescence signal, the performance or specificity of sgRNA is preliminarily verified. The terminal point of fluorescence value curve of target amplification product and negative control should be significantly different.

[0068] The calculation formula of fluorescence intensity growth rate is as follows:

[0069]

[0070] Wherein, Fluorescence Slope represents the fluorescence growth rate in the application, Rn represents the fluorescence signal of the nth minute (the fluorescence signal of the 15th minute is used in the application), and R0 represents the background fluorescence signal.

[0071] In the application, the fluorescence intensity of the amplification product of each sgRNA and the corresponding template is determined by CRISPR reaction. The fluorescence curve and the fluorescence growth rate trend are shown in Figure 2 The analysis result shows that the DV target DV-sgRNA-3 has better effect, and can be used for subsequent RPA primer screening.

[0072] 2.3, primer design and verification

[0073] 2.3.1 RPA primer design

[0074] In the detection target range, select 30-35 base length fragments as RPA primer candidates; in order to ensure stability and specificity, the GC content of RPA primer should be between 40%-60%, and the RPA amplification fragment is usually 100-200 bp. According to the primer design principle, 3 upstream and downstream primers are designed for RPA primer verification. The RPA primers (primer position see Figure 3 ) shown in Table 6 are designed for each template for subsequent synthesis and verification experiments.

[0075] Table 6 RPA primer table

[0076]

[0077] 2.3.2 RPA primer screening

[0078] DV-sgRNA-3 with good performance in the previous verification is used for RPA primer screening, and the specific screening steps are as follows:

[0079] 1) Mix the isothermal amplification freeze-dried ball with 48 μL amplification buffer A thoroughly;

[0080] 2) Take 12µL of the liquid after dissolving the freeze-dried ball and place it on the eight-tube cap, and add 1.6 µL of nucleic acid template;

[0081] 3) Take a new row of eight-tube, respectively add 0.2µL of upstream primer and 0.2µL of downstream primer (primer concentration 100µM);

[0082] 4) Add 1.0µL of magnesium acetate to the reaction tube, and for multiple reactions, it is recommended to add magnesium acetate to the inside of the reaction tube in step 3, and slowly cover the eight-tube cap in step 2 to avoid liquid falling inside the cap, and do not mix yet;

[0083] 5) Configure 10µL of CRISPR system according to the AaCas12b protein instruction, and add the configured CRISPR system to the new eight-tube cap;

[0084] 6) Place the eight-tube cap with the cap in step 4 on the centrifuge, and centrifuge for a moment to make all components fall to the bottom of the tube, vortex for 10s, and mix thoroughly;

[0085] 7) Discard the eight-tube cap in step 6, replace it with the eight-tube cap with the CRISPR system, avoid the CRISPR system falling into the tube, do not centrifuge, and then immediately place the reaction tube into the constant temperature device for incubation at 43℃ for 30min;

[0086] 8) After the RPA process is completed, centrifuge for a moment to mix the RPA system with the CRISPR system, vortex for 10s, mix thoroughly, and then place it in an instrument that can read fluorescence, incubate at 43℃ for 30min, and read fluorescence every 1min.

[0087] The results of the DV target primer screening are as follows: the upstream primers DV-RPA-F1, DV-RPA-F2, DV-RPA-F3 and the downstream primers DV-RPA-R1, DV-RPA-R2, DV-RPA-R3 in the application are used for cross primer performance verification (see Table 6 for sequences). According to the primer verification results, the DV-RPA-F2 / R1 primer group has better detection performance at a lower concentration (see Figure 4 ). Therefore, DV-RPA-F2 / R1 is used in the subsequent performance verification of the DV target RPA-CRISPR detection system in the application with DV-sgRNA-3.

[0088] The plasmid copy number calculation formula in the application is:

[0089] In the formula, N plasmid is the calculated plasmid copy number concentration, unit copies / µL; NA Avogadro's number, 6.02E+23; C is the concentration of the plasmid after determination, in ng / μL; N is the number of base insertions in the plasmid, 2710 is the fragment length of the puc57 vector, and 660 is the average molecular weight of base pairs. The present application calculates the copy number concentration of the plasmid at a specified concentration according to the above formula, and dilutes to a specific concentration for different verifications. i Avogadro's number, 6.02E+23; C is the concentration of the plasmid after determination, in ng / μL; N is the number of base insertions in the plasmid, 2710 is the fragment length of the puc57 vector, and 660 is the average molecular weight of base pairs. The present application calculates the copy number concentration of the plasmid at a specified concentration according to the above formula, and dilutes to a specific concentration for different verifications.

[0090] 2.4, detection system verification

[0091] 2.4.1, sensitivity verification of detection system

[0092] The plasmid template is diluted downward, and the gradient setting includes 1E3 copies / T, 1E2 copies / T, 1E1 copies / T, 1E0 copies / T, and 1E-1 copies / T. The primers and sgRNA combination determined in 2.3.2, and the same CRISPR system (Table 5) are used to verify the sensitivity of the target. Each gradient is verified by 3 experimental repeats.

[0093] According to the sensitivity verification results (Table 6) Figure 5 ), and combined with the difference significance analysis, it is preliminarily determined that the DV target can detect 1E0 copies / T of sample per reaction.

[0094] After completing the sensitivity verification of each target, the present application continues to perform 10 experimental repeats near the detection limit of each target to confirm the sensitivity of the target. According to the sensitivity of each target verified in the early stage, the present application selects 1E1 and 5E0 copies / T concentration groups of DV target for sensitivity confirmation research. The amplification curves and fluorescence signal growth rates of the detection limit verification of each target are shown in Table 7 Figure 6 .

[0095] 2.4.2, real sample verification

[0096] Based on the completion of the verification of the detection system of the DV target, the present application continues to verify the actual sample of the detection system. The sample used in this part of the verification is 16 real samples of nucleic acid provided by the customer for verification, and the detection method in SN / T 2301-2009 is used for qPCR gold standard control (see Figure 8 ), and the detailed sample information is shown in Table 7. The reaction system and primer sgRNA combination used in the test are the same as those in 2.3.2. The detailed verification results are shown in Table 8 Figure 7 and Table 8. The real sample verification results show that all the sample detection results are positive, which is consistent with the detection results of qPCR.

[0097] Table 7 Actual sample information

[0098]

[0099] Table 8 Comparison of actual sample detection results and qPCR detection results

[0100]

[0101] 2.4.4, specificity verification

[0102] The present application uses 4 cross samples provided by the customer (2019.JK-11 (serum), JK-45, WD133F V2, WD133F V3, see 7 for details) to test specificity, with sample nucleic acid 2 (type I dg2-157) as a positive control, cross species as a specificity verification test group, and water as a negative control template. The primers and sgRNA combinations determined in 2.3.2, as well as the same CRISPR system, were used to verify the specificity of the target. Each cross reaction was verified twice.

[0103] In the present application, the results of specificity verification of DV target are shown in the figure Figure 9 The specificity verification results show that the RPA-CRISPR detection method developed in the present application for DV target can only get positive amplification in target DV (sample nucleic acid 2 (type I dg2-157)), and the detection results of the remaining 4 cross species are negative, indicating that the detection method developed in the present application for DV target has good specificity.

[0104] Example 2 SynsorPocket-One platform detection system development and verification

[0105] 1. SynsorPocket-One platform chip preparation process

[0106] Due to the particularity of SynsorPocket-One platform, in-situ verification of CRISPR system is required before verification. The CRISPR in-situ system of the present application is shown in Table 9.

[0107] Table 9 SynsorPocket-One process CRISPR detection system preparation

[0108]

[0109] After the above CRISPR system is prepared, 30 μL of the system is added to a SynsorPocket-One platform detection hole, and the chip is placed in a 50°C oven, and the system is dried for 1.5 h; after drying is completed, the chip is taken out, and after being covered with a film, it is placed in a sealed aluminum foil bag and stored in a low-humidity environment. Before use, the reagent protection cover should be unscrewed, and the chip protection cover above the sample addition port should be removed.

[0110] 2. SynsorPocket-One platform detection process

[0111] In this part of the test, first, the RPA amplification system of the target is prepared, and the addition amount of the RPA freeze-dried ball in the system is 2. The addition amount and the final concentration of each component in the RPA system in this process are shown in Table 10.

[0112] Table 10 SynsorPocket-One platform RPA system preparation

[0113]

[0114] After mixing all the reaction systems in RPA, all the systems are transferred to the SynsorPocket-One chip, and then the convex part of the detection reagent card is completely pressed down, and the sample liquid is completely filled in the detection hole of the detection reagent card. According to the direction indicated in the hand-held area of the detection reagent kit, the chip is inserted into the instrument detection card port. Finally, press the instrument start button, at this time the Test indicator light should be in the normal state, after the indicator light area is lighted (the longest detection time of the Pocket platform system is 50 min), the experimental result reading is carried out. At the same time, the platform can be connected to a computer to obtain and analyze real-time fluorescence data.

[0115] 3. Fluorescence threshold verification of SynsorPocket-One platform

[0116] In order to ensure that the SynsorPocket-One platform can stably convert the amplification fluorescence signal into the output positive and negative signal, the present application first carries out the detection of the fluorescence threshold of the platform. In this part of the study, the test of the blank sample of the platform LOB is determined by testing the blank sample under different instruments, different operators, different times, different environments, and based on the statistical summary of the LOB values at different time points, the threshold values at different time points are determined combined with the average value of the LOB of different experimental times and the standard deviation value of the LOB. The threshold value is integrated into the platform, so as to determine the positive and negative results of the test.

[0117] 4. SynsorPocket-One platform result analysis

[0118] Based on the test results of the Pocket platform, the positive and negative judgment is as followsFigure 10 As shown: positive (+): the Synsor Pocket-One platform system can output a positive result in the shortest 10 min according to the algorithm inside the instrument, at this time, the Positive red light on the instrument is always on, and the detection result is positive; negative (-): after the Synsor Pocket-One platform completes all detection processes, if the fluorescence still does not exceed the threshold line, a negative result will be output, at this time, the Negative green light on the instrument is always on, and the detection result is negative; invalid: when the instrument or chip is abnormal, the Error red light on the instrument is always on, the detection result is invalid, and the instrument and chip need to be replaced and re-operated.

[0119] 5. Synsor Pocket-One platform feasibility verification

[0120] On the basis of the early RPA-CRISPR process establishment, the present application intends to first verify the feasibility of the Synsor Pocket-One platform, and uses 1E4 copy / T target samples and cross samples for preliminary verification, and verification is carried out under the Synsor Pocket-One platform detection process. The test results are shown in Figure 11 .

[0121] 6. Synsor Pocket-One platform sensitivity results

[0122] The plasmid sample is diluted by 10 times gradient, and the gradient is set to 1E3 copy / T, 1E2 copy / T, 1E1 copy / T, 1E0 copy / T. The primers and sgRNA combination determined in 2.3, and the Synsor Pocket-One platform detection process are used to verify the sensitivity of the target. In the present application, the sensitivity verification results are shown in Figure 12 , the preliminary sensitivity test results show that the RPA-CRISPR Synsor Pocket-One platform detection system in the present application can realize the detection of 1E2 copy / T samples.

[0123] Obviously, the above-mentioned specific embodiments only further detail the purpose, technical scheme and beneficial effects of the present application, and it should be understood that the above-mentioned is only a specific example of the present application and is not used to limit the present application, any modification, equivalent replacement, improvement, etc. made within the spirit and principles of the present application should be included in the protection scope of the present application.

Claims

1. A dengue virus RPA-CRISPR / Cas12b detection system, comprising an RPA amplification reaction system and a Cas12b detection reaction system, wherein the Cas12b detection reaction system comprises an sgRNA probe, characterized in that, The RPA amplification reaction system includes RPA primer pairs with sequences as shown in SEQ ID NO. 4~5, and the nucleotide sequence of the sgRNA probe is shown in SEQ ID NO.

6.

2. A reagent kit for visually detecting dengue virus, characterized in that, It includes an sgRNA probe and RPA primer pairs as shown in SEQ ID NO. 4-5; the nucleotide sequence of the sgRNA probe is shown in SEQ ID NO.

6.

3. The reagent kit for visually detecting dengue virus according to claim 2, characterized in that, The kit also includes AaCas12b enzyme protein, 10×AaCas12b Buffer, ssDNA reporter molecule, and nuclease-free water; the ssDNA is FAM-TTTTTTT-BQ1.

4. The use of the kit according to claim 2 in the preparation of dengue virus detection reagents.

Images